1. Field of the Invention
This invention relates generally to a solution particularly useful for supporting the in vitro growth of, and preservation of vascular endothelial cells. More particularly, the invention comprises a solution which can support ex vivo or in situ preservation without extreme hypothermia of organs, tissues, and explants.
2. Description of the Background and Related Art
A) Vascular Endothelial Cell Culture
The blood vessel wall architecture consists of three major components: the adventitia, the media, and the intima. The intima, or the lumenal area, consists of a confluent monolayer of vascular endothelial cells with tight junctions which function as a permeability barrier. It was once generally assumed that the vascular endothelial cell population was homogeneous throughout the vasculature. It is now recognized that vascular endothelial cells are a heterogeneous cell population with wide variation in morphology, function, antigen expression, and growth requirements. The history of the tissue culture of vascular endothelial cells began approximately 20 years ago when human umbilical vein endothelial cells were first cultured (Simionescu, N, Simionescu: Histology, Edited by L. Weiss, McGraw-Hill Book Co., 1977, pg. 373; Jaffe et al., J. Clin. Invest. 52:2757, 1973). Subsequent to the work with umbilical cells, endothelial cells isolated from large vessels such as aorta, saphenous vein, and vena cava, have been successfully supported in cultured by a medium such as that disclosed by Levine in U.S. Pat. Nos. 4,994,387 and 5,132,223. Vascular endothelial cells from microvessels, i.e. capillaries, arterioles, and venules, are important to studies of inflammation and neovascularization. However, microvasculature endothelial cells could be maintained in culture, but could not be passaged (Booyse et al., Thromb. Diath. Haemorrh. 34:825, 1975).
Since a limited number of vascular endothelial cells can be isolated from a blood vessel or a piece of tissue, typically incubation in tissue culture is required to expand the cell population or to enrich the purity of the cell preparation for laboratory procedures requiring rather large populations of vascular endothelial cells. Traditionally, the tissue culture of vascular endothelial cells relied on the use of a basal medium such as RPMI-1640 or Medium 199, supplemented with serum, heparin, and endothelial cell growth factors (Thorton et al., Science 22:623, 1983). The serum in this media presumably supplies the necessary nutrients, hormones, and attachment factors lacking in the basal media. A basal media which can be used without serum supplementation or minimal supplementation with serum may be desirable because of the economic benefits from reducing serum usage. The heparin, which is produced by most mammalian cells in tissue culture, potentiates the growth promoted by the endothelial cell growth factors. Endothelial cell growth factor is required to provide the positive signal to initiate vigorous cell replication. Heparin sulfate is the only mucopolysaccharide to date which has been recognized to potentiate the activity of growth factors in endothelial cells. It has been recognized since 1956 that heparin-like materials were involved in the process of cell division. However, very little biochemical evidence exists to identify the exact physiologic role of heparin sulfate. There is some discrepancy in the literature as to how effective heparin supplementation is in supporting the growth of microvessel endothelial cells. Heparin and dextran sulfate have been shown to increase bovine capillary endothelial cell migration. However, neither heparin nor dextran sulfate has any effect on capillary endothelial cell proliferation (Azizkhan et al., J. Exp. Med. 152:931, 1980; Zetter, Diabetes 30:24, 1981). In contrast, in other studies, human capillary endothelial cells have been maintained in long term culture with heparin sulfate supplementation (Jarrell et al., J. Vas. Surg. 1(b):758, 1984). While other mucopoly-saccharides have been previously tested for their ability to support the growth of vascular endothelial cells, to date only heparin sulfate has been recognized and used for this purpose (Folkman et al.: Cold Spring Harbor, Cell Proliferation, 1982).
Primary cultures of microvessel endothelial cells are fragile. If contaminating cell types, most notably capillary pericytes, are eliminated, the endothelial cells can be maintained undisturbed, and passage is now possible (Goetz et al., In Vitro Cell Dev. Biol. 21:172, 1985). By using isolation techniques which enrich the microvessel endothelial cell population and with optimizing culture conditions, it has been reported that endothelial cells can be maintained for up to 12 passages with cloned capillary endothelial cells, and for only 5 passages for microvessel cells, i.e. venules and arterioles (Booyse et al., Thyromb. Diath. Haemorrh. 34:825, 1975; Goetz et al., In Vitro Cell Dev. Biol. 21:172, 1985; Kern et al., J. Clin. Invest. 71:1822, 1983; Folkman et al., Textbook of Rheumatology (ed. Wm Kelley), Vol.1, pg.210, WB Saunders). However, this is substantially less cell growth than can be obtained with human umbilical vein endothelial cells. The cultivation of other fastidious cell types is even more difficult. If other cell types, most notably isolated cells of the kidney, explants, and whole tissues, are not transfected or immortalized, the period of time they can be viably maintained in vitro is quite short.
Therefore, there exists a need for a preservation solution as a tissue culture medium which has the ability to support the attachment and cultivation of vascular endothelial cells from a variety of anatomic sites. A desirable feature of such a solution is that the formulation can support the growth of large vessel and microvessel endothelial cells simultaneously, as well as being useful in the cultivation of a variety of other fastidious cell types, most notably the isolated cells of the kidney.
Different media formulations have been disclosed in the art which contain one or more components contained in the tissue culture medium of the present invention. However, none of the media formulations known in the art disclosed a) the combination of components of the tissue culture medium of the present invention, which b) support the simultaneous growth of large vessel endothelial cells and microvessel endothelial cells of c) human, murine, bovine, porcine, canine and rat origin from d) various anatomical sites including kidney, heart, brain, aorta, vena cava, and fat-drived.
Further, it is known to those skilled in the art that a basal medium used for the growth and support of, for example, a transformed cell will be different than the formulation for growth of fastidious cells, i.e. large vessel and microvessel endothelial cells. A comparison of the formulation of the present invention with representative formulations known in the art is summarized in Table 1.
TABLE 1 __________________________________________________________________________ present parameter invention Ref. 1 Ref. 2 Ref. 3 Ref. 4 Ref. 5 __________________________________________________________________________ cell HUVEC; rat HUVEC; porcine murine human type VEC; pulmon- VEC corneal mammary lym- micro- ary VEC tumor pho- vessel EC micro- blast vessel line EC basal disclosed DMEM & M199 .TM. EMEM DMEM & RITC medium in Table HF12 with HF12 56-1 2 (1:1) Earle (1:1) or salts 80-7 growth retinal- pitui- ECGF EGF none none factor derived tary acidic ex- FGF* tract & ECG MPS heparin; none hepa- CDS none none CDS rin Mg sup- 1.24 g/L 1 g/L none none none none plement Serum &lt;1% FBS, 2-10% 20% 20% FBS BSA/ BSA/ protein BSA FBS FBS oleic fatty acid/ acid .alpha.CD & .alpha.CD growth: 17 hrs. none 17-21 39 hrs. none 24 dt stated hrs. stated hrs. for HUVEC growth: 3 days none 6 days 9-16 none 7 tc stated days stated days growth: none vitro- gela- none fibro- none matrix (untreat- gen tin nectin supple- ed) ment growth: Factor Factor Factor not N/A, N/A, cp VIII.sup.+, VIII.sup.+, VIII.sup.+, stated not VEC not ACE.sup.+, diL- ACE.sup.+ VEC diL-Ac- Ac-LDL.sup.- LDL.sup.+ __________________________________________________________________________ Ref. 1: Tanswell et al. (1991, J. Dev. Physiol. 15:199-209) Ref. 2: Levine et al. (U.S. Pat. No. 4,994,387) Ref. 3: Lee et al. (1991, Kaoshiung J Med Sci 7:614-621) Ref. 4: Kawamura et al. (1985, Dokkyo J. Med. Sci. 12:167-180) Ref. 5: Yamane et al. (1981, Proc. Japan Acad. 57:385-389) * retinal derived acidic FGF (fibroblast growth factor) commercially available as ENDO GRO .TM. (VEC TEC Inc.) Abbreviations: HUVEC human umbilical cord vein endothelial cells; VEC large vessel vascular endothelial cells; EC endothelial cells; DMEM & HF12 Dulbecco's modified Eagle's medium and Ham's F12 medium; EMEM Eagle's minimal essential medium; ECG endothelial cell growth supplement; ECGF endothelial cell growth factor; EGF epidermal growth factor; MPS mucopolysaccharide; Mg magnesium containing compound; BSA bovine serum albumin; FBS fetal bovine serum; .alpha.CD alphacyclodextrin; dt population doubling time; tc time to confluence; cp conservation of phenotype; ACE angiotensinconverting enzyme, expressed when the cells are confluent in culture; diLAc-LDL- the ability to take up acetylated low density lipoprotein; N/A not applicable.
B) Organ Transplantation
Organ transplantation is a highly public therapeutic modality. Unlike any other medical innovation, transplantation has caught and maintained the public's interest. In most cases transplantation is the therapy of choice for end-stage organ disease.
Living-related donor organ donation has steadily increased because of the severe organ shortage, living-related donation increased by 7% in 1990 alone. The size of the transplant waiting list grew by 37% between 1988 and 1990. During the same period, the number of organ donors grow by only 9%.
There are three major problem areas in organ donation/procurement:
1) limited organ donor pool--Most organs in the United States are procured from heartbeating cadavers; i.e., patients who succumb to head trauma and are therefore, brain dead and are maintained on life support systems. Heartbeating cadavers represent a small factor of trauma patients and therefore, represent a limited organ donor pool.
2) obtaining consent--When families are not confronted with the decision of terminating life-support systems, these families are much more likely to make the decision to donate, as in the case of corneas. Therefore, opening new avenues of organ donation where there would not be a decision to terminate life-support systems will not only provide a new source of organs, but in all probability would also increase the current rate.
3) preservation and associated problems--Why can't organs be harvested from non-heartbeating cadavers? The moment an organ donor's heart stops beating, the cessation of blood flow results in ischemia. The onset of ischemia initiates a phase of metabolic depression leading to cell death. We know that within 60 minutes, warm ischemia will lead to the necrosis of the proximal convoluted tubules. The historic approach to organ preservation involves hypothermia. The reduction of tissue temperature results in a lower metabolic activity. However, hypothermia preservation is not benign; it results in edema, alterations in permeability, and tubule damage. The principal difference between ischemia at warm and cold temperatures is the rate at which the cell injury and death occur. Therefore, warm ischemia damage represents the major obstacle to expanding the organ donor pool into the non-heartbeating cadaver population. Organs damaged by warm ischemia cannot tolerate further damage incurred by hypothermia. Until the damaging effects of ischemia can be alleviated, the donor pool cannot be expanded.
There are seven major parameters involved in the in vitro preservation of organs: 1) ischemia, 2) the effects of the mandatory hypothermia, oxygen consumption in hypothermically preserved organs, 4) ATP synthesis, 5) ion pumps, 6) alterations in permeability leading to edema and 7) reperfusion injury.
1) Ischemia
Ischemia, or the cessation of blood flow, will cause the phenomenon of no reflow, which is the failure of the circulation to return. Ischemia-mediated damage is most severe in the first and third segments of the proximal convoluted tubules and this damage is directly related to the length of ischemia. The initial effects of ischemia are from the lack of molecular oxygen for oxidative phosphorylation; which leads to the depletion of ATP stores within the mitochondria. Nucleotides are rapidly lost during ischemia and this loss is an important factor in the failure of tissue subjected to prolonged ischemia to regenerate ATP after the restoration of the blood supply.
2) Hypothermia
Currently, all preservation technology is dependent upon hypothermia to diminish the effects of ischemia. The benefits of hypothermia were recognized early on, when in 1937 Bickford and Winton noted that hypothermia prolonged the duration of tissue survival. Hypothermia exerts its beneficial effect by diminishing the oxygen demand of the organs and also by reducing the metabolic rate. Normal oxygen consumption by the kidney is high, approximately 6.3 ml/min. This oxygen consumption is reduced to about half at 30.degree. C. and to less than 5% at 4.degree. C. Most organs are stored at temperatures ranging from 4.degree.-10.degree. C. Similarly, below 22.degree. C. a cessation of glomerular filtration occurs and below 18.degree. C. tubular activity is inhibited. Most enzyme systems functioning at normothermia show an approximately two-fold decrease for every ten degrees decrease in temperature.
However, the side-effects of hypothermia are not benign. Cold-induced damage entails organ swelling, loss of endothelial cell integrity, acute tubular necrosis (ATN), inhibition of the ion pumps and intracellular acidosis. In fact, hypothermia may be the rate-limiting factor in organ preservation. To control this cold-induced damage, all clinical perfusates employ a variety of impermeants and colloids to control cell swelling.
3) Oxygen Consumption
Supplying adequate oxygen delivery to the organs was a major obstacle to success in early studies of organ preservation. Oxygen consumption in the kidney is high and this oxygen consumption correlates with renal transport processes.
Hypothermia, while reducing the rate of metabolism and oxygen consumption, also blocks the effective utilization of oxygen by tissues. At normal physiologic temperatures, the phospholipids making up the cell membranes are highly fluid. Under the hypothermic conditions utilized in organ preservation, the lipid bilayer experiences a phase-change and becomes gel-like, with greatly reduced fluidity. This essentially frozen lipid in the cell membranes negates the utilization of oxygen, even in the presence of a high oxygen-tension. Without the required oxygen, the metabolic consequence for preserved organs is glycolysis.
4) ATP
Most ATP is synthesized in mitochondria via oxidative phosphorylation. The mitochondria utilize oxygen and substrate to convert ADP to ATP and in the process reduce oxygen to H.sub.2 O. This controlled reduction requires the addition of four electrons. The cytochrome oxidase complex accomplishes this in one step. In doing the reduction in one step, toxic free radical intermediates are not generated.
Ischemia, whether warm or cold, initiates a rapid fall in cellular ATP levels. ATP can be readily resynthesized from adenosine once oxidative phosphorylation resumes at normothermia. Without oxidative phosphorylation, glycolysis is the only means of producing ATP. However, glycolysis is twenty times less efficient than oxidative phosphorylation. The salvage pathway of ATP production produces reactive oxygen species in the process of metabolizing hypoxanthine to xanthine and xanthine to uric acid by means of xanthine dehydrogenase. These toxic free radical intermediates include the superoxide anion radical, hydrogen peroxide and hydroxyl radical. Mitochondria normally maintain efficient control systems generating minimal levels of free radicals. Scavengers, which effectively reduce the small amount of these intermediates generated under normal conditions, abound in vivo.
The depletion of ATP causes a shutdown of the sodium pump, active Ca.sup.++ extrusion stops, fatty acid accumulates and degraded phospholipids are not regenerated. Acidosis develops because the protons released during the synthesis of ATP cannot be converted to H.sub.2 O by normal oxidative metabolism.
3) Ion Pumps
The major impact of ATP depletion is the shutdown of the ion pumps, in particular, the sodium pump. The sodium pump is responsible for maintaining the intracellular balance of sodium and potassium and for normal cell volume regulation. The pump exchanges sodium for external potassium. The lack of ATP to drive the pumps results in increased intracellular sodium, more than there being a fall in potassium. The vascular endothelial cells can then swell to double their thickness very quickly. This swelling leads to alterations in permeability resulting in leaky endothelium. If the supply of energy is reestablished before the death of the cells occurs, the process can be reversed and cell volume returns to normal.
6) Edema
Therefore, the preservation of membrane integrity is probably the major fundamental issue to organ preservation. In all cases where metabolism is inhibited, the result is edema due to increased intracellular H.sub.2 O content. The development of leaky endothelium leads to a reduction in blood flow in the medulla which leads to a secondary necrosis of the tubules, which then leads to obstruction and a reduction of glomerular filtration, urine flow and urine concentrating capacity. Therefore, the damage to the endothelium plays a major role in the subsequent renal damage secondary to the preservation.
7) Reperfusion Injury
The overall effect of hypothermic preservation is tissue hypoxia. Cold preservation followed by rewarming leads to reperfusion injury. Reperfusion injury following hypothermia is a well established concept and its main focus in on the endothelium. Toxic free radical intermediates initiate an injury cascade involving cellular derangement, leukocyte/platelet adhesion and hypercoagulation. Various scavengers and agents have been used such as superoxide dismutase (SOD) and catalase. Calcium antagonists such as chlorpromazine and prostacyclin and its analog have been used with varying degrees of success. Obviously, it is more important to avoid the generation of these radicals rather than to attempt to eliminate them.
It is apparent that the degree of preservation/reperfusion injury is the direct result of the duration of the cold preservation, and not the reperfusion, since reperfusion after short periods of cold ischemia does not lead to graft injury. The extent of free radical production is related to the length of the cold preservation. Likewise, blood cell adhesion is directly related to the preservation damage.
Hypothermia is the essential foundation of current technology used in organ preservation. All recent progress in organ preservation can be traced directly to maneuvers used to control the very damage caused by the hypothermia itself; namely using impermeants and colloids to control cell swelling, pharmacologic agents to stop nucleotide waste, and to limit reperfusion injury while maintaining the membrane integrity.
For example, the third generation of perfusates, most notably the UW solution or VIASPAN.TM. (Belzer et al., Transpl. 33:322-323, 1986), are totally synthetic solutions devoid of all animal protein. VIASPAN.TM. uses HES to avoid toxicity. There are eleven ingredients in the VIASPAN.TM. solution:
______________________________________ phosphate buffer to prevent acidosis adenosine a precursor for ATP synthesis, it also has vasodilating properties and is a platelet inhibitor magnesium cofactor for cation-dependent events allopurinol xanthine oxidase inhibitor to block oxygen radical production glutathione to assist in handling oxidative stress and for its reducing capabilities during lipid peroxidation, which may be important during reperfusion HES a colloid to prevent expansion of the extracellular space raffinose provides osmotic support lactobionate a major organic impermeant anion, since it does not permeate the membrane and therefore, prevents cell swelling ions it is also an intracellular-like solution, high in potassium. ______________________________________
It is of interest to note that replacing potassium ions with sodium ions in the VIASPAN.TM. solution, does not affect the quality of the preservation and some reports describe improved results, particularly in liver transplantation. VIASPAN.TM. is superior to previous perfusates and generally represents state-of-the-art organ preservation. However, many researchers have questioned the effectiveness of some of ingredients. There is general agreement that the lactobionate is required, while only one study found HES to be required.
There are now several offshoots of the VIASPAN.TM. solution, including the HTK, HP16, and Cardisol solutions using haemacel or PEG to replace the HES and other sugars to replace the raffinose and impermeants to replace the lactobionate. Today we now have the situation where a new perfusate's efficacy is usually compared to VIASPAN.TM..
The Future
Certainly it is clear that the existing organ donor pool must somehow be expanded. If we are to expand the donor base into the nonheartbeating cadaver population, a different approach to organ preservation is needed. Warm ischemic damage represents the major obstacle to utilizing nonheartbeating cadavers and similarly, warm ischemically damaged organs cannot tolerate a second insult of hypothermic damage. Interestingly, many of the preservation related problems of severe hypothermia would be eliminated at a more moderate level of hypothermia. Future preservation ("warm preservation") may be in the range of 18.degree.-35.degree. C., where membrane lipids are in a more normal state. Almost everything that occurs at 37.degree. C. also occurs at 20.degree. C., but at a slower rate. More moderate hypothermia would help to: prevent toxic free radicals rather than using scavengers at the time of reperfusion, eliminate vasospasm, support oxygen utilization and raise the metabolic rate during preservation. Concordant with using nonheartbeating cadavers, there will be a need to develop in vitro parameters of graft viability. And there will probably be a need of organ specific perfusates, designed to support a higher level of metabolism during warm preservation techniques.
Therefore, there is a need for a preservation solution useful for initial organ flushing and as a perfusate for in situ or ex vivo preservation of organs for transplantation using a warm preservation technology which minimizes, or even repairs, damage due to warm ischemia, and which supports the organ near normal metabolic rate. A desirable feature of using such a solution is that organ preservation may be extended further by increasing metabolic activity, by eliminating severe hypothermia, and supplying adequate oxygen and metabolite delivery to support this basal metabolism.